Considerable progress has been made in the past years in reducing malaria morbidity and mortality in Africa, with Cameroon inclusive, largely due to interventions such as LLIN and use of artemisinin-based combination therapy [21, 31]. Detailed assessments of parasite carriage by conventional diagnostics alongside molecular investigation have uncovered that a considerable proportion of malaria infections is undetected by routine microscopy [32]. In settings where recent malaria control efforts have been successful, and across various endemicities, sub-microscopic infections frequently outnumber microscopically detectable infections [5, 16, 33]. Although high levels of asymptomatic and sub-microscopic infection occur in many different settings [19, 33–35], studies on their clinical significance are still lacking. This cross-sectional study examines the influence of asymptomatic and sub-microscopic P. falciparum infection on anaemia and haematological indices as public health problems in children ≤ 14 years across low, middle belt and highland altitudes in the Mount Cameroon area.
Findings from the study suggests that children < 5years and 5–9 years in the middle belt and lowland respectively, are the most affected by the malaria parasite and therefore constitute sensitive groups for monitoring changes in malaria burden using microscopy in the Mount Cameroon area. Case management which is one of the current surveillance methods in the country focus more on the < 5 years age group, although asymptomatic malaria parasite which is also higher among the 5–9 years age group may greatly contribute to transmission. Consequently, health education and treatment should not only target vulnerable groups (< 5 years and pregnant women) but all the age groups.
The prevalence and density of asymptomatic malaria parasitaemia with respect to age and sex were significantly different across the different altitudinal sites. This result is not surprising because several studies have reported that in Cameroon, malaria burden and transmission intensity are heterogeneous with spatial and temporal variations between altitudes and geographical areas, with prevalence rates varying from one area to another [4]. Although the prevalence of asymptomatic malaria parasite was comparable between males and females, the density was however higher in males than in females among middle belt dwellers. This is in line with an earlier study by Kimbi et al. [36] and Sumbele et al. [21]. In addition, the effect of sex on the outcome of P. falciparum infection has previously been reported in other parts of Africa [37, 38]. Hormonal differences between the sexes may also be a contributing factor to the difference in malaria parasite prevalence. Cernetich et al. [39] showed that synthesis of testosterone by males suppresses antiplasmodial immune response, whereas production of oestrogen augments antiplasmodial immune response.
The present study is the first large-scale description of sub-microscopic malaria parasite prevalence among children in three communities in the Mount Cameroon area using the nested PCR method. The overall sub-microscopic malaria parasitaemia of 43.8% was observed in microscopic negative slides by PCR in the study population. In line with other studies, Okell et al. [15] reported that the proportion of sub-microscopic infections is inversely correlated with slide prevalence and parasite density on the global level. Bousema et al. [5] reported that individuals with sub-microscopic malaria parasite are accountable for maintaining Plasmodium species between transmission season, since they are a major reservoir.
Findings from the study indicated that the proportion of sub-microscopic infection in the communities were significantly associated with age, as older children had an increased chance of being carriers of sub-microscopic infection compared with those younger. This is consistent with reports from other studies from Uganda [40], Kenya [41], India [42] and Ethiopia [43] who reported that older children do not easily develop symptomatic malaria upon infection both because they tolerate parasite densities better without developing fever and because they are at lower risk to develop high parasite densities. Age is a key factor that correlates positively with protective immunity in malaria-endemic areas. It has been reported that parasitaemia in older age groups is lower than the detection limits of conventional malaria diagnostic tools, which therefore fail to detect parasitaemia [44].
The importance of the sub-microscopic parasite pool rests on the understanding that sub-microscopic infections can transmit malaria [45], although the minimum parasite density necessary for transmission is unknown. Worthy of note in the Mount Cameroon area, sub-microscopic Plasmodium infections in older children may be an important source of the local transmission of the parasite. In addition, the unusual significantly higher GMPD observed in children < 10 years living in the highlands than those in the middle belt is also of concern especially as the climatic conditions in the highlands are considered unfavourable for the development of the vector and transmission of the parasite.
The prevalence of sub-microscopic infection was highest in the highland dwellers than lowland with children in the highlands having a lower malaria prevalence by microscopy when compared with their lowland counterparts. This observation support findings of other studies [14, 46] which suggests that the burden of sub-microscopic infections is highly heterogeneous across different locations. Although several hypotheses could account for this, one possible explanation might be differences in the extent of parasite genetic diversity between settings [47–49]. In low transmission settings, repeated exposure to a limited number of strains might lead to rapid development of protective immunity against those strains. Individuals in these settings would then be expected to have, on average, a higher proportion of infected sub-microscopic population. By contrast, in high transmission settings, higher circulating parasite genetic diversity would mean that individuals are more frequently infected with strains they have not previously encountered. However, in contrast, a recent characterisation of sub-microscopic malaria carriage at three Ugandan sites with varied transmission intensity revealed little change in the extent and size of the sub-microscopic reservoir across the transmission gradient at the sub-national level [13].
In addition to altitudinal effect that may affect transmission dynamics, findings from the study revealed non-users of ITN were 2 folds more likely to carry sub-microscopic infection. This observation supports the findings of other studies that proper use of ITN significantly reduces malaria morbidity and mortality [2, 50].
It has been reported that sub-microscopic and asymptomatic infections go undetected and untreated with little or no clinical manifestation in many malaria endemic communities [51]. However, findings from this study demonstrated that these infections are associated with anaemia as well a decrease in some red cell indices and platelet counts. Anaemic children were 9 times more likely to carry sub-microscopic infection when compared to non-anaemic children, demonstrating the clinical relevance of sub-microscopic infection. The result agrees with studies by Rek et al. [13] and De Mast et al. [35] who also suggested an association between sub-microscopic malaria infection and anaemia. Anaemia is multifactorial and observations from this study enriches the body of evidence suggesting the detrimental clinical consequences of parasitaemia of any density [17, 33].
The high prevalence of anaemia (62.3%) in children less than or equal to 14 years among the population in this area highlights anaemia as a severe public health problem in malaria endemic communities. The association between malaria parasitaemia and anaemia is well established in previous studies [26, 36, 52–54]. Malaria parasitaemia causes devastation of parasitized and non-parasitized red blood cells hence reducing haemoglobin levels leading to anaemia. The higher proportion of anaemia in the younger age group is in line with previous studies that anaemia due to malaria is more severe in younger children in areas of intense transmission [55, 56]. Children in this age group are more vulnerable to infection with malaria than others with severe and potentially fatal complications.
Sub-microscopic Plasmodium infection in the study was associated with lower Hb, Hct, RBC count as well as MCHC as confirmed by the decrease in their mean values in those positive. It is most likely that sub-microscopic Plasmodium infection would have exacerbated the reduction in the red cell indices as asymptomatic parasitaemia and protracted malaria infections have been associated with a marked reduction in Hb concentration and with a clinically significant RBC destruction [57] indicating that parasitological cure is necessary for haematological recovery [58].
Findings revealed reduction in platelet count in children with sub-microscopic Plasmodium infection. The association of platelet count and malaria has previously been described [59]. However, the reduction did not culminate in thrombocytopaenia which is the reduction in platelet count below the normal range that has been postulated as a marker of Plasmodium infection. Thrombocytopaenia seems to occur through peripheral destruction [60], excessive removal of platelet by spleen pooling [61] as well as platelet consumption by the process of disseminated intravascular coagulopathy. Also, immune-mediated destruction of circulating platelets has been postulated as a cause of thrombocytopaenia [62].
While the findings reported have implications for the control and elimination of malaria in the Mount Cameroon area it could have a wider applicability in other regions with similar altitudinal ranges and environmental conditions. The study is however not without limitation, the study design does not allow the assessment of causality between sub microscopic parasitaemia and anaemia.